PROJECT SUMMARY: Acute Myeloid Leukemia (AML) remains a highly fatal disease due to the development of drug resistance during chemotherapy. The tumor microenvironment, in particular the crosstalk between leukemia cells and bone marrow mesenchymal stromal cells, has been implicated to be critical for both cancer development and drug resistance. We recently identified a large number of differentially regulated genes in bulk RNA-sequencing of healthy and AML stroma that potentially support the survival of leukemic cells within the bone marrow microenvironment. However, it is unknown if these transcripts are universally up-regulated in stromal cells or if certain pre-existing subpopulations expand and predominate in the marrow microenvironment to support the survival of leukemia cells. A corollary of this is that evaluation of bulk stromal cells by itself is insufficient to fully explain the process and mechanism by which stromal cells mediate AML drug resistance. As such, the overarching goal of this project is to define the pathological stromal landscape that drives AML drug resistance. I will test the underlying hypothesis that specific mesenchymal stromal cell subpopulations within the microenvironment contribute to drug resistance and enable survival of AML cells throughout the course of therapy. This hypothesis will be tested by performing single-cell RNA-sequencing on AML stroma from patients (1) before treatment, (2) on-treatment, and (3) following the development of drug resistance. Imaging and mechanistic studies will also be performed to spatially localize prioritized subpopulations and investigate the molecular mechanisms that confer drug resistance over time. These data will enable me to identify specific stromal subsets that are dynamically regulated during the development of resistance and change spatially with respect to the location of leukemic cells within the microenvironment. Moreover, these data will provide insight into how these subpopulations alter the AML microenvironment, leading to protection of leukemia cells and, thereby AML relapse. The specific aims of this proposal are as follows: (1) Define specific subsets of mesenchymal stromal cells that correlate with the development of AML drug resistance, and (2) Determine the mechanism by which proteins expressed by stromal cells confer drug resistance within the AML microenvironment. Results from this proposal have the potential to create a paradigm shift in the treatment of AML, emphasizing the need to consider extrinsic mechanisms of resistance that are equally critical in understanding clinical resistance and determining disease outcomes.